Regulatory T cell epitope and hepatitis C virus homolog

ABSTRACT

A pharmaceutical composition used to suppress immunity in an animal, wherein the pharmaceutical composition comprises an isolated T-cell epitope peptide containing an amino acid sequence of PLLLLLLXLPXRA (SEQ ID NO: 5), wherein X is an amino acid.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from U.S. Provisional Application Ser.No. 61/899,617 filed Nov. 4, 2013, the entire content and substance ofwhich is incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 6, 2015, isnamed 9842_SL.txt and is 1,959 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AI082642awarded by National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Artificial induction of tolerance to self or to foreign antigens is thegoal of therapy for autoimmunity, transplantation allergy and otherdiseases, and is also desirable in the context of therapy withautologous proteins and non-autologous proteins. Until recently,therapeutic tolerance induction relied on broad-based approaches thatresulted in cellular depletion and cytokine profile alteration. Thesebroad-based approaches weaken the immune system in general and leavemany subjects vulnerable to opportunistic infections, autoimmune attackand cancer. There is a need in the art for less aggressive and moretargeted approaches to the induction of immune tolerance.

Immune tolerance is regulated by a complex interplay between T cells, Bcells, cytokines and surface receptors. Initial self/non-selfdiscrimination occurs in the thymus during neonatal development wheremedullary epithelial cells express specific self protein epitopes toimmature T cells. T cells recognizing self antigens with high affinityare deleted, but autoreactive T cells with moderate affinity sometimesavoid deletion and can be converted to so called ‘natural’ regulatory Tcells (T_(reg)) cells. These natural T_(reg) cells are exported to theperiphery and provide for constant suppression of autoimmunity.

A second form of tolerance occurs in the periphery where mature T cellsare converted to an ‘adaptive’ T_(reg) phenotype upon activation viatheir T cell receptor in the presence of IL-10 and TGF-.beta. Thepossible roles for these ‘adaptive’ T_(reg) cells include dampeningimmune response following the successful clearance of an invadingpathogen as a means of controlling excessive inflammation as might becaused by an allergic reaction or low level chronic infection, orpossibly to facilitate co-existence with beneficial symbiotic bacteriaand viruses. ‘Adaptive’ T_(reg) may also play a role in managing thelife cycle of human antibodies that have undergone somatichypermutation.

Natural regulatory T cells are a critical component of immune regulationin the periphery. Upon activation through their TCR natural Tregs arecapable of suppressing bystander effector T cell responses to unrelatedantigens through contact dependent and independent mechanisms. Inaddition the cytokines released by these cells including IL-10 andTGF-.beta., are capable of inducing antigen-specific adaptive Tregs.Despite extensive efforts, with few exceptions, the antigen specificityof natural Tregs, and more importantly natural T_(reg) circulating inclinically significant volumes, is still unknown.

There is need in the art for the identification of new regulatory T cellepitopes contained in common autologous proteins such as IgG(“Tregitopes”) and for methods for related to their preparation and ofuse.

Chronic hepatitis C virus (HCV) infection is a major public healthconcern worldwide. It is the leading cause of liver failure and reasonfor liver transplant in the US. Irrespective of clinical outcome, acuteHCV infections are characterized by broad HCV-specific T cell responsesthat correlate with spontaneous viral clearance in a minority ofindividuals. In most patients, however, this initial response fails tocontain the virus and chronic disease results. Increased numbers of CD4⁺regulatory T_((reg)) cells circulating in the bloodstream andaccumulating in the liver have been implicated in the pathogenesis ofchronic hepatitis C.

T_(reg) cells constitute one of the major mechanisms underlyingimmunological homeostasis and self-tolerance. In addition, T_(reg) cellsplay a key role in moderating the immune response to infectiousdiseases, suppressing host tissue and organ damage that would occur inthe absence of regulation. Although key to maintaining immunehomeostasis, a growing body of evidence suggests that T_(reg) cells alsocontribute to the establishment and persistence of chronic infections,e.g., HCV. While no single marker exists, human T_(reg) cells areclassically identified by expression of the transcription factor,forkhead box P3 (FoxP3), and the cell surface expression of theinterleukin (IL)-2 receptor a chain (CD25). Bystander suppression is anadditional key feature of T_(reg) cells.

Two distinct T_(reg) cell subsets are described in the literature:natural (n)T_(reg) cells specific for self epitopes and generated byhigh-avidity selection in the thymus, and inducible (i)T_(reg) cellsthat derive from conventional (CD4⁺CD25⁻FoxP3⁻) T cells followingstimulation in the periphery. nT_(reg) cells can induce the conversionof conventional T cells to iT_(reg) cells via cytokine-dependent and-independent mechanisms, a process called infectious tolerance. Notably,the factors that affect expansion of the T_(reg) cell population incases of chronic hepatitis C remain to be fully delineated. Nonetheless,the consensus supports the heterogeneous nature of the expanded T_(reg)cell population composed of both nT_(reg) and iT_(reg) cell subsets.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A: the production of CD3+CD4+FoxP3+ cells in cultures of PBMCsderived from HCV-infected patients for the medium alone.

FIG. 1B: HCV GI_p7_794 induces the production of CD3+CD4+FoxP3+ cells incultures of PBMCs derived from HCV-infected patients.

FIG. 1C: the production of CD3+CD4+FoxP3+ cells in cultures of PBMCsderived from HCV-infected patients for HCV G1_NS4b_1941.

FIG. 1D: the production of CD4 cells shown in FIG. 1C, further showingamounts of C25 and C349.

FIG. 2: HCV_G1_p7_794 fails to elicit a significant increase mCD3+CD4+FoxP3+ cells in PBMC cultures derived from non-infectedindividuals or patients who spontaneously cleared infection.

FIG. 3: CD3+CD4+FoxP3+ T cells do not proliferate m response toHCV_G1_p7_794.

FIG. 4: Human p7_794 analog stimulates a significant increase mCD3+CD4+FoxP3+ T cells in HCV-infected and non-infected individuals.

FIG. 5: Fewer HCV-G1_p7_794-responsive Treg cells express CD304(neuropilin).

SUMMARY OF THE INVENTION

The present invention harnesses the functions of regulatory T cells(T_(reg)), particularly those cells that already regulate immuneresponses to foreign and self proteins in the periphery (pre-existing ornatural T_(reg)). In one aspect, the invention provides T-cell epitopepolypeptide compositions.

The selective engagement and activation of pre-existing natural Tregthrough the use of Tregitopes and Tregitope-antigen fusions, istherapeutically valuable as a means of treatment for any disease orcondition marked by the presence of an unwanted immune response createdby autoimmune diseases, such as, but not limited to, Crohn's Disease,Guillian Barre Syndrome, Lupus, Psoriasis, Rheumatoid Arthritis,Ulcerative Colitis and Multiple Sclerosis; pre- or post-transplanations;allergies such as, but not limited to, asthma, COPD and allergicrhinitis. The present invention is directed to the use of a peptide inthe HCV p7 protein, HCV_G1_p7_794 (SEQ ID NO: 1), residing in thehepatitis C virus (HCV), for the control of unwanted immune response. Afurther embodiment is the human analog (SEQ ID NO: 2) of theHCV_G1_p7_794, H p7_794.

In one embodiment, the present invention is directed to a T-cell epitopepolypeptide composition comprising at least one polypeptide selectedfrom the group consisting of: SEQ ID NOS: 1 and 2. In a particularembodiment, the invention is directed to a pharmaceutical compositioncomprising a polypeptide of the invention and a pharmaceuticallyacceptable carrier.

In a second embodiment, the present invention is directed to a nucleicacid encoding at least one T-cell epitope polypeptide selected from thegroup consisting of: SEQ ID NOS: 1 and 2. In a particular embodiment,the invention is directed to a vector comprising a nucleic acid of theinvention. In yet another embodiment, the invention is directed to acell comprising a vector of the invention.

In a third embodiment, the invention is directed to a method of treatingor preventing a medical condition in a subject in need thereofcomprising administering a therapeutically effective amount of a T-cellepitope polypeptide selected from the group consisting of: SEQ ID NOS: 1and 2. In a particular embodiment, the medical condition is selectedfrom the group consisting of: an allergy, an autoimmune disease, atransplant related disorder, graft versus host disease, an enzyme orprotein deficiency disorder, a hemostatic disorder, cancer, infertility;and a viral, bacterial or parasitic infection.

In fourth embodiment, the present invention is directed to a method forrepressing immune response in a subject, comprising administering acomposition comprising a therapeutically effective amount of a peptidecomprising SEQ ID NOS: 1 or 2 to the subject, wherein the peptiderepresses the immune response. In a particular embodiment, the peptidesuppresses effector T cell response. In a particular embodiment, thepeptide suppresses helper T cell response. In another embodiment, thepeptide suppresses B cell response.

In a fifth embodiment, the present invention is directed to a method ofsuppressing antigen specific immune response in a subject through theadministration of a therapeutically effective amount of a compositioncomprising SEQ ID NOS: 1 or 2, wherein the one or more Tregitopes areeither covalently bound, non-covalently bound or in admixture with aspecific target antigen resulting in the diminution of immune responseagainst the target antigen. In a particular embodiment, the suppressiveeffect is mediated by natural Treg. In another embodiment, thesuppressive effect is mediated by viral homolog of the natural Treg. Inanother embodiment, the peptide suppresses effector T cell response. Inanother embodiment, the peptide suppresses helper T cell response. Inanother embodiment, the peptide suppresses B cell response.

In a sixth embodiment, the present invention is directed to a method forenhancing the immunogenicity of a vaccine delivery vector, comprisingidentification and removal of regulatory T cell epitopes residing in thevaccine to hepatitis C virus. In a particular embodiment, the T cellepitopes are selected from the group consisting of: SEQ ID NOS: 1 or 2.In a further embodiment, a vaccine delivery vector with removedregulatory T cell epitopes is further enhanced, comprising (a) isolatingregulatory T-cells from the biological sample; (b) contacting theisolated regulatory T-cells with an effective amount of a Tregitopecomposition of the enhanced vaccine delivery vector; (c) identificationof the sequences; and (d) removal of remaining regulatory T cellepitopes residing in the vaccine.

In a seventh embodiment, the present invention is directed to a methodto reduce the repressing immune response in a subject infected with thehepatitis C virus, comprising administering a therapeutically effectiveof the antibody recognizing a peptide from the group consisting of: SEQID NOS: 1 and 2.

DETAILED DESCRIPTION OF INVENTION

Spontaneous resolution of hepatitis C virus (HCV) infections dependsupon a broad T cell response to multiple viral epitopes. Most patientsfail to clear infections spontaneously, however, and develop chronicdisease. The elevated number and function of CD3⁺CD4⁺CD25⁺FoxP3⁺regulatory T_((reg)) cells in HCV-infected patients suggest the role ofT_(reg) cells in impaired viral clearance. Factors contributing toincreased T_(reg) cell activity in chronic hepatitis C cases remain tobe delineated.

Resolution of primary HCV infections is dependent upon the vigorousresponse of CD4⁺ and CD8⁺ T cells to multiple viral epitopes. HCVpersists in the majority of infected patients, however, by modifyingand/or evading the host immune response. Purportedly, a variety offactors contribute to the diminished T cell responses observed inchronically infected patients including: viral mutation and escapelinked to both CD4 and CD8 T cell failure, CD4 T cell anergy, CD8 T cellexhaustion, impaired dendritic cell function, and T_(reg) cell-mediatedsuppression. The increased frequency of T_(reg) cells found in the liverand circulating in the peripheral blood of chronically-infected patientsprovided an initial indication of the role of T_(reg) cells in thepathogenesis of chronic hepatitis C. It remained unclear until recently,however, whether this increase represented the HCV epitope-specificresponse of T_(reg) cells or the nonspecific consequence of chronicinflammation and liver disease.

Immunoinformatics tools were used to predict promiscuous,highly-conserved HLA-DRB1-restricted immunogenic consensus sequences(ICS), each composed of 5-6 T cell epitopes. These sequences weresynthesized and added to cultures of peripheral blood mononuclear cells(PBMCs) derived from patients who resolved HCV infection spontaneously,patients with persistent infection, and non-infected individuals.

In the present invention, surprisingly a unique viral peptide derivedfrom HCV p7 protein (HCV_G1_p7_794) was identified that promotes aT_(reg) cell response among PBMCs derived from patients with persistentHCV infection. This peptide exhibited human homology when evaluatedusing GenBank Basic Local Alignment Search Tool (BLAST). Furtheranalysis using a new bioinformatics tool, JanusMatrix, demonstrated thatthis HCV peptide cross-reacts with HLA matched peptide sequences locatedwithin hundreds of human proteins. Our invention showed thatHCV_G1_p7_794 engages preexisting nT_(reg) cells, as a consequence ofthis homology, induces infectious tolerance and the expansion aniT_(reg) cell population, which contributes to suppression of effectorT_((eff)) cell activity in cases of chronic HCV infection. Further, avirus-encoded peptide (HCV_G1_p7_794) with extensive human homologyactivates cross-reactive CD3⁺CD4⁺CD25⁺FoxP3⁺ nT_(reg) cells,contributing to immunosuppression and chronic hepatitis C.

The invention provides methods of treating a subject with a medicalcondition comprising administering a therapeutically effective amount ofa T cell epitope selected from the group consisting of: SEQ ID NOS: 1 or2 in a pharmaceutically acceptable carrier or excipient. The T cellepitope of the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. The pharmaceuticalcompositions generally comprise at least one T cell epitope and apharmaceutically-acceptable carrier in a form suitable foradministration to a subject. Pharmaceutically-acceptable carriers aredetermined in part by the particular composition being administered, aswell as by the particular method used to administer the composition.

Example 1: a Peptide Sequence in HCV p7 Exhibits Human Homology

Sustained, virus-specific CD4 and CD8 T cell responses are associatedwith successful control of HCV infection. Therapeutic vaccination offersa rational approach to stimulating host resistance and overcoming viralpersistent in cases of chronic disease. Toward this end, 20 promiscuousHCV (genotype 1) ICS were predicted and validated by demonstrating theirability to bind a panel of the eight common alleles HLA-DRB1 alleles:DRB1*0101, *0301, *0401, *0701, *0801, *1101, *1301, and *1501,representing essentially the entire human population. One ICS, locatedwithin HCV p7 protein (HCV_G1_p7_794; WPLLLLLLALPQRAYAQ (SEQ ID NO: 1)),exhibited significant human homology (>70% shared identities) determinedby GenBank BLAST analysis; none of the remaining ICS exhibited the samehomology.

HCV sequences were acquired from the Los Alamos sequence and immunologydatabase. Nine-mer amino acid sequences, capable of fitting the bindinggroove of HLA class II molecules and highly conserved across HCVgenotype 1a and 1b isolates, were identified using bioinformatics tools.Each 9-mer was scored for its predicted potential to bind a panel ofeight HLA class II alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701,DRB1*0801, DRB1*1101, DRB1*1301 and DRB1*1501).

HLA class II immunogenic consensus sequences (ICS) were constructed byassembling potentially immunogenic 9-mers into 18-25 amino acidsequences. ICS construction improves the probability that an epitopewill be presented in the context of more than one HLA allele, thusbroadening the response of an HLA-diverse human population. Twenty ICSwere synthesized as peptides using 9-fluoronylmethoxycarbonyl chemistryand purified >85% by 21st Century Biochemicals (Marlboro, Mass.). Aspredicted, each of these “promiscuous” ICS was bound by multipleHLA-DRB1 alleles in competitive binding assays performed in accordancewith methods we described previously. Each ICS was also evaluated forhuman homology (>7 shared identities per 9-mer frame) using GenBankBLAST.

Example 2: HCV_G1_p7_794 Induces a CD3⁺CD4⁺FoxP3⁺ T Cell Response inHCV-Infected Patients

HCV-seropositive subjects with persistent viremia (Ab⁺VL⁺), patients whospontaneously resolved infection (Ab⁺VL⁻) and HCV-seronegative,non-infected (Ab⁻VL⁻) individuals were recruited from the Rhode IslandAdult Corrections Institution (ACI) to participate in this study.Recruitment methods and the population are described elsewhere. TheInstitutional Review Boards of the Miriam Hospital, Rhode IslandDepartment of Corrections and Office of Human Research Protectionapproved this study. All HCV infected subjects included in theseanalyses were infected with HCV genotype 1a or 1b (Versant HCV genotypeassay 2, Siemens Healthcare Diagnostics Inc.). Hartford HospitalTransplantation Research Laboratory (Hartford, Conn.) and the CellularMediated Immunology Core Laboratory at the University of Rhode Island(Providence, R.I.) performed HLA typing. Subject demographic andserologic data are shown in Table 1.

TABLE 1 Viral Load Geno- Anti- ID# Age HCV Status IU/ml type bodyHLA-DRB1 229 28 Infected 170,620 1 + 03,11 236 29 Infected 602,611 1 +01,11 261 25 Infected 7,464,611 1 + 01,11 265 22 Infected 21,917,190 1a + 13,15 272 23 Infected 458  1a + 03,11 279 25 Infected 1,788,326 1a + 13,15 282 28 Infected 186,678  1a + 03,04 216 23 Clearer NA NA +07:01 221 27 Clearer NA NA + 03,11 222 25 Clearer NA NA + 03,15 246 26Clearer NA NA + 01,04 267 23 Clearer NA NA + 03,11 264 27 Clearer NANA + 03,11 248 26 Non-infected NA NA − 04,11 259 26 Non-infected NA NA −04,11 262 22 Non-infected NA NA − 03,11 257 22 Non-infected NA NA −03,15 263 26 Non-infected NA NA − 01,03 268 21 Non-infected NA NA −

A series of experiments was undertaken to determine and compare theHCV_G1_p7_794-specific responses of PBMCs obtained from non-infectedcontrol individuals (Ab⁻VL⁻), patients who spontaneously cleared HCVinfection (Ab⁺VL⁻) and infected patients in whom viremia persisted(Ab⁺VL⁺).

Cryopreserved PBMCs were thawed; suspended in HEPES-buffered RPMI1640medium supplemented with 10% HuAB serum (Valley Biomedical, Winchester,Va.), glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin; andrested overnight at 37° C. in a humidified, CO₂ incubator. On thefollowing day, the PBMCs were centrifuged and resuspended in freshmedium containing 5% HuAB serum; 1×10⁶ cells/nil were transferred todeep, flat-bottom, non-treated 48-well plates. The peptide sequenceindicated in the text was added (10 μg/ml final concentration) and thecells were incubated for 5 days. PBMCs cultured in the presence ofmedium with 0.1% DMSO served as a negative control. Half the spentmedium was replaced on day 3. The cells were collected for analysis onday 5.

For proliferation assays, cells rested overnight were labeled withcarboxyfluorescein diacetate, succinimidyl ester (CFSE: LifeTechnologies Corporation, Carlsbad, Calif.) prior to culture in thepresence or absence of 30 ng/ml anti-CD3 monoclonal antibody (cloneHIT3a; BioLegend, San Diego, Calif.) and 10 μg/ml HCV_G1_p7_794. Cellproliferation was evaluated by flow cytometric analysis according to theprotocol provided by Life Technologies in which a loss in fluorescenceintensity correlates directly with the extent of replication.

The cells collected after 5 days incubation were quantified andcharacterized in accordance with methods we described previously.Dye-conjugated mouse monoclonal antibodies specific for the followingdeterminants were purchased from BioLegend (San Diego, Calif.) and used:CD3, CD4 (clone OKT4), CD8a (clone HIT8a); CD39 (clone A1), and CD304(clone 14H4); anti-human CD25 (clone M-A251) and anti-human FoxP3 (clone236a) were purchased BD Biosciences (San Jose, Calif.). Data werecollected on a 4 laser (13 color capability) BD LSRII Flow Cytometer (BDBiosciences, San Jose, Calif.) and analyzed using FlowJo software (TreeStar, Inc., Ashland, Oreg.). All analyses were conducted using theappropriate isotype controls to correct for non-specific staining PBMCswere cultured in the presence of medium alone, 10 μg/ml HCV_G1_p7_794,or 10 μg/ml HCV_G1_NS4b_1941.

The results were analyzed using the SigmaStat statistics program (AspireSoftware International). Individual means were compared using anon-paired Student's t test or a Mann-Whitney Rank Sum test. Dataderived from 3 or more groups were compared by one-way analysis ofvariance; the Dunnett's test was used to determine which groups differedsignificantly.

The cells were collected after 5 days incubation and analyzed by flowcytometry (FIGS. 1A-1C). The CD3+CD4+FoxP3+ cells were furthercharacterized by the expression of CD25 and CD39 (FIG. 1B). The gatingscheme and representative analyses are shown.

HCV_G1_p7_794 induced a marked increase in CD3+CD4+FoxP3+ cells whenadded to PBMC cultures derived from infected, Ab+vL+ patients (FIGS.1A-1C). In contrast, HCV_NS4B_1941 (AARVTQILSSL TITQLLKRLHQWI; an ICSthat exhibits little human homology) failed to promote an increase inCD3+CD4+FoxP3+ cells when added to PBMC cultures derived from Ab+vL+individuals. Other highly conserved HCV ICS with negligible humanhomology (e.g., HCV_G1_NS4b_1769; ISGIQYLAGLSTLPGNPA) similarly failedto elicit a CD3+CD4+FoxP3+ cell response (data not shown). TheCD3+CD4+FoxP3+ cells induced by the addition of HCV_G1_p7_794 to Ab+vL+PBMC cultures expressed both CD25 (IL-2 receptor a chain, constituentlyexpressed by Treg cells) and CD39, a cell ectonucleotidase associatedwith Treg cell suppressor function (FIG. 1D).

Ab⁺VL⁺ patients also had a higher baseline level of CD3⁺CD4⁺Foxp3⁺ cellscompared to spontaneously clearers and non-infected controls, a findingconsistent with the literature (FIG. 2). PBMCs obtained from infectedpatients (Ab⁺VL⁺, n=4), patients who clear infection (Ab⁺VL⁻, n=6) andnon-infected controls (Ab⁻VL⁻, n=4) were cultured in the presence ofmedium alone, 10 μg/ml HCV_G1_p7_794 or 10 μg/ml HCV_G1_NS4b_1941. Thecells were collected after 5 days incubation, stained and analyzed byflow cytometry as outlined schematically in FIG. 1; *Significantlydifferent, P=0.014; **significantly greater than all other groups,P<0.001.

Further, neither HCV_G1_p7_794 (putative Treg epitope) norHCV_G1_NSB4_1941 (putative T effector epitope) added to PBMC culturesderived from non-infected individuals or from patients who successfullycleared HCV infection induced a significant increase in CD3⁺CD4⁺FoxP3⁺cells.

Example 3. HCV_G1_p7_794 Induces an Increase in CD3⁺CD4⁺FoxP3⁺ T Cellsin the Absence of Proliferation

The hallmark of CD3⁺CD4⁺FoxP3⁺ T_(reg) cells is suppressor activity.CSFE-labeled, Ab⁺VL⁺ PBMCs were cultured with medium alone or mediumthat contained 30 ng/ml anti-CD3, 10 μg/ml HCV_G1_p7_794 or acombination of anti-CD3 and HCV_G1_p7_794. After 5 days incubation, thecells were collected and proliferation the total (FIG. 3BA) and CD3⁺CD4⁺(FIG. 3B) PBMC populations was estimated by flow cytometry. Data wereobtained in a single experiment representative of the results obtainedfor PBMCs derived from six HCV-infected patients.

CD3⁺CD4⁺FoxP3⁺ T cells do not proliferate in response to HCV_G1_p7_794.PBMCs obtained from HCV-infected patients (Ab⁺VL⁺, n=4) and non-infectedcontrols (Ab⁻VL⁻, n=4) were cultured in the presence or absence of 10μg/ml of human p7_794 analog. The cells were collected after 5 daysincubation and analyzed by flow. Significantly more CD3⁺CD4⁺FoxP3⁺ cellswere recovered from PBMC cultured with the human p7_794 analog thanmedium alone: *P=0.001; **P=0.048.

The addition of HCV_G1_p7_794 to Ab⁺VL⁺ PBMC cultures resulted in areproducible, albeit slight, decrease in the proliferative response toanti-CD3 monoclonal antibody treatment whether the total (FIG. 3A) orCD3⁺CD4⁺ (FIG. 3B) cell population was assessed. Importantly, theaddition of HCV_G1_p7_794 alone (i.e., in the absence of anti-CD3) hadno effect on the proliferation of CD3⁺CD4⁺ T cells obtained from thesame patient population. This finding suggests that HCV_G1_p7_794induces the conversion of conventional CD3⁺CD4⁺FoxP3⁻ T cells toCD3⁺CD4⁺FoxP3⁺ iT_(reg) cells, rather than stimulating the proliferationof T_(reg) cells already present.

Example 4: HCV_G1_p7_794 Activates Cross-Reactive nT_(reg) Cells andInduces Infectious Tolerance

A human analog of HCV_G1_p7_794 (p7_794, PLLLLLLSLPPRA (SEQ ID NO: 2))was identified by GenBank BLAST analysis and synthesized in an effort toprovide a clearer understanding of the nature of the T_(reg) cells thatrespond to HCV_G1_p7_794. Like the HCV-encoded homolog, the human analoginduced a significant increase in CD3⁺CD4⁺FoxP3⁺ cells in PBMC culturesderived from patients with persistent viremia (FIG. 4).

Human p7_794 analog stimulated a significant increase in CD3⁺CD4⁺FoxP3⁺T cells in HCV-infected and non-infected individuals. PBMCs obtainedfrom HCV-infected patients (Ab⁺VL⁺, n=4) and non-infected controls(Ab⁻VL⁻, n=4) were cultured in the presence or absence of 10 μg/ml ofhuman p7_794 analog. The cells were collected after 5 days incubationand analyzed by flow. Significantly more CD3⁺CD4⁺FoxP3⁺ cells wererecovered from PBMC cultured with the human p7_794 analog than mediumalone: *P=0.001; **P=0.048.

In contrast to HCV_G1_p7_794, the human analog also induced anapproximate three-fold increase in CD3⁺CD4⁺FoxP3⁺ cells in PBMC culturesderived from non-infected individuals indicating the response of annT_(reg) cell population.

CD304 (neuropilin-1) is expressed by a subset of FoxP3⁺ T_(reg) cells inhumans. In mice, CD304 expression differentiates natural (CD304⁺), frominducible (CD304⁻), T_(reg) cells. PBMCs obtained from an infectedpatient (representative of 4 patients) were incubated in medium alone(A) or medium that contained 10 μg/ml HCV_G1_p7_794 (B). The cells werecollected on day 5, stained and analyzed by flow. Panels on the rightindicate the percentage of CD4⁺FoxP3⁺ cells in each population thatexpresses CD304. While a similar distinction has yet to be reported inhumans, it is pertinent to note that the bulk of CD4⁺FoxP3⁺ cellscontained among Ab⁺VL⁺ PBMCs cultured in the absence of HCV_G1_p7_794expressed CD304 indicative of nT_(reg) cells (FIG. 5A). In contrast, thevast majority of CD4⁺FoxP3⁺ cells among Ab⁺VL⁺ PBMCs cultured in thepresence of HCV_G1_p7_794 were CD304-negative characteristic of iT_(reg)cells (FIG. 5B). Together these findings showed that HCV_G1_p7_794recognition by nT_(reg) cells following HCV exposure promotes infectioustolerance and the production of FoxP3⁺ iT_(reg) cells from conventionalCD4⁺FoxP3⁻ T_(eff) cells.

Example 5: JanusMatrix Confirms T_(reg) Cell Epitopes Shared byHCV_G1_p7_974 and the Human Proteome

By comparing HCV G1_p7_794 with peptide sequences found within the humanproteome, JanusMatrix analysis provided further insight into thecapacity of HCV_G1_p7_794 to induce a T_(reg) cell response by PBMCsderived from HCV-infected patients.

Crystal structure analyses of ternary, MHC:epitope: T cell receptor(TcR) complexes indicate that certain amino acid residues of a T cellepitope contact the MHC molecule while other residues contact the TcR.The TcR contacts can be modeled using a new bioinformatics tool,JanusMatrix. This tool interrogates potential T cell epitopes from bothits HLA-binding and TcR-facing aspects, and assesses TcRcross-reactivity with T cell epitopes that are present in the humangenome and in the human microbiome, or other genomes. Those epitopesfrom two different genomic sources, e.g., HCV and human, that bind thesame HLA molecules and present identical amino acids to the TcR aredesignated potentially cross-reactive, as they may stimulate the sameTcR and trigger the same T cell to respond. In the analysis, JanusMatrixdivided the HCV HLA-DRB1-restricted epitopes (comprising the ICSdescribed above) into TcR-facing and MHC-binding amino acid residues.The human protein database (UniProtKB) was searched for TcR-facingepitopes that cross-react with epitopes encoded by HCV.

As illustrated in Table 2, HCV_G1_p7_794 consists of 6 T cell epitopes,5 of which cross-react with 152 putative human T cell epitopes containedin 264 different human proteins. Similarly, the human analog, p7_794,cross-reacts with putative T cell epitopes located within severalhundred human proteins. On the other hand, neither HCV_G1_NS4b_1941(control ICS often used in the ex vivo experiments described above) norany of the other 18 ICS, which were originally predicted and validated(data not shown), exhibited significant cross-reactivity with the humanproteome. The results of these analyses support the speculation thatHCV_G1_p7_794 activates a cross-reactive nT_(reg) cell population thatnormally functions to suppress autoimmune responses to a large number ofhuman proteins, which contain a common peptide sequence (epitope).

TABLE 2 Comparison of Peptide Sequences # Cross-Reactive Epitopes perin Human Description ICS Sequence ICS Proteins HCV_G1_p7_794WPLLLLLLALPQ 5.0 264 RAYAQ SEQ ID NO: 1 Human p7_794 PLLLLLLSLPPR 4.0325 A SEQ ID NO: 2 HCV_G1_NS4b_1941 AARVTQILSSLT 6.0  28 ITQLLKRLHQWISEQ ID NO: 3

The present invention demonstrated that ICS, HCV_G1_p7_794, induced amarked increase in T_(reg) cells in PBMC cultures derived from infectedpatients, but not those patients who spontaneously cleared HCV ornon-infected individuals. An analogous human peptide (p7_794), on theother hand, induced a significant increase in T_(reg) cells among PBMCsderived from both HCV infected and non-infected individuals. JanusMatrixanalyses determined that HCV_G1_p7_794 is comprised of T_(reg) cellepitopes that exhibit extensive cross-reactivity with the humanproteome.

This unique viral peptide derived from HCV p7 protein (HCV_G1_p7_794)promoted a T_(reg) cell response among PBMCs derived from patients withpersistent HCV infection. It also exhibited human homology whenevaluated using GenBank Basic Local Alignment Search Tool (BLAST).Further analysis using a new bioinformatics tool, JanusMatrix,demonstrated that this HCV peptide cross-reacts with HLA matched peptidesequences located within hundreds of human proteins. The datademonstrated that HCV_G1_p7_794 engaged preexisting nT_(reg) cells, as aconsequence of this homology, induces infectious tolerance and theexpansion an iT_(reg) cell population, which contributes to suppressionof effector T_((eff)) cell activity in cases of chronic HCV infection.It is concluded that HCV_G1_p7_794 with extensive human homologyactivates cross-reactive CD3⁺CD4⁺CD25⁺FoxP3⁺ nT_(reg) cells,contributing potentially to immunosuppression and chronic hepatitis C.

The ability of HCV-derived epitopes to stimulate T_(reg) cell responsesis well documented; a number of HCV-encoded T_(reg) cell epitopesderived from structural, as well as non-structural, HCV proteins havebeen reported. The invention described is the first to identify apromiscuous, HCV peptide sequence (HCV_G1_p7_794) that exhibitsextensive human homology and the ability to induce T_(reg) cells invitro. HCV_G1_p7_794 added to PBMCs cultures derived from HCV-infectedpatients, but not from non-infected individuals or patients who clearedinfection, induced a marked increase in CD3⁺CD4⁺FoxP3⁺ cells. Inaddition to expressing CD25, characteristic of T_(reg) cells, the vastmajority of these cells expressed CD39, a marker that distinguishesFoxP3⁺ T_(reg) cells from activated T_(eff) cells that transientlyexpress FoxP3, but not suppressor activity.

In contrast to HCV_G1_p7_794, the human peptide analog (p7_794) eliciteda significant increase in CD3⁺CD4⁺FoxP3⁺ cells in PBMC cultures derivedfrom non-infected individuals with no evidence of prior HCV exposure, aswell as HCV-infected patients. This finding is congruent with thesuggestion that viral epitopes with human homology influence thepathogenesis of chronic HCV by activating preexisting, cross-reactivenT_(reg) cells. Indeed, extensive homology between the HCV polyproteinand proteins that comprise human proteome is well documented.JanusMatrix, a bioinformatics algorithm that interrogates potential Tcell epitopes from both their HLA-binding and TcR-facing aspects,confirmed the existence of significant homology between HCV_G1_p7_794and hundreds of proteins that compose the human proteome. The results ofthis analysis demonstrate the potential efficacy of JanusMatrix inidentifying pathogen-encoded epitopes that elicit the activity ofnT_(reg) cells, which normally function to suppress autoimmunereactivity to self antigens (proteins). In this regard, it is pertinentto remark that HCV_G1_p7_794 is comprised of epitopes that arehomologous to those found in hundreds of human proteins. This suggeststhe autoimmune response to a large number of proteins is inhibited by asingle or limited number of nT_(reg) cell clones responsive to a commonpeptide sequence, rather than a large number of clones each responsiveto a unique sequence in each protein.

Although immunosuppression is a defining characteristic of T_(reg) cellsand readily demonstrated in animal (mouse) models, demonstrating thesuppressor activity of human CD3⁺CD4⁺CD25⁺FoxP3⁺ T_(reg) cells in vitro,however, has proven problematic. Thus, while HCV_G1_p7_794 induced a 3-to 4-fold increase in CD3⁺CD4⁺CD25⁺FoxP3⁺ cells in PBMC cultures derivedfrom HCV-infected patients in the studies reported here, these cellsexerted only a limited effect on the nonspecific proliferative responseof cells stimulated with anti-CD3. Recent studies indicate that thenature of the responder T cells (CD4⁺CD25⁻ versus CD4⁺CD25^(low)) andthe ratio of purified T_(reg) cells to purified responder cells exertsignificant effects on the outcome of suppression assays. Notably, inthe experiments reported here, HCV_G1_p7_794 was added to heterogeneousPBMC cultures in which the CD3⁺CD4⁺FoxP3⁻ responder, far outnumbered theCD3⁺CD4⁺CD25⁺FoxP3⁺ suppressor, cells by >10:1. Importantly, theaddition of HCV_G1_p7_794 alone to PBMC cultures derived fromHCV-infected (Ab⁺VL⁺) patients failed to induce cell proliferationdespite a marked (3- to 4-fold) increase in CD3⁺CD4⁺CD25⁺FoxP3⁺ cellnumber. This finding suggests that HCV_G1_p7_794 induces the conversionof conventional T_(eff) to T_(reg) cells, i.e., infectious tolerance, asuggestion supported by studies demonstrating the inability of nT_(reg)cells to proliferate in response to their cognate antigen in vitro.Furthermore, in contrast to Ab⁺VL⁺ PBMCs cultured in medium alone, onlya minority of CD3⁺CD4⁺FoxP3⁺ cells derived from PBMCs cultured in thepresence of HCV_G1_p7_794 expressed CD304 (neuropilin), which isexpressed by a subset of FoxP3⁺ T_(reg) cells in humans and associatedspecifically with nT_(reg) cells in mice. While it has been suggestedalternatively that the expanded T_(reg) cell population in chronic, HCVinfected patients is composed of cells phenotypically similar tonT_(reg) or iT_(reg) cells, our results concur with the consensus thatthe expanded T_(reg) cell population in chronic HCV-infected patients isheterogeneous, composed of both T_(reg) cell subsets.

Taken together, the findings showed that HCV non-structural protein p7contains a unique peptide sequence (HCV_G1_p7_794), which is recognizedby the TcR repertoire expressed by nT_(reg) cells that function normallyto suppress the autoimmune response to hundreds of human proteins. UponHCV_G1_p7_794 recognition, these nT_(reg) cells induce the conversion ofconventional T_(eff) cells to iT_(reg) cells (i.e. infectioustolerance). It is expected that the nT_(reg) cells and iT_(reg) cellsresponsive to HCV_G1_p7_794 contribute to the elevated T_(reg) cellpopulation found in HCV-infected patients, and play a role inimmunosuppression and viral persistence.

Those skilled in the art will appreciate that numerous modifications andvariations may be made to the above disclosed embodiments and containedin the claims without departing from the spirit and scope of the presentinvention.

What is claimed is:
 1. A pharmaceutical composition comprising anisolated T-cell epitope peptide adapted to repress an immune responseand a pharmaceutically acceptable carrier or excipient; said peptideconsists of an amino acid sequence of PLLLLLLXLPXRA (SEQ ID NO:5),wherein X is an amino acid and does not have to be the same amino acidin each occurrence in a given sequence.
 2. The pharmaceuticalcomposition of claim 1, wherein the PLLLLLLXLPXRA peptide is selectedfrom the group consisting of PLLLLLLSLPPRA (SEQ ID NO:2) andPLLLLLLALPQRA (SEQ ID NO:6).
 3. The pharmaceutical composition of claim1, further comprising a target antigen.
 4. An isolated T-cell epitopepeptide, consisting of an amino acid sequence of PLLLLLLXLPXRA (SEQ IDNO:5), wherein X is an amino acid and does not have to be the same aminoacid in each occurrence in a given sequence.
 5. The T-cell epitopepeptide of claim 4, wherein the PLLLLLLXLPXRA sequence is selected fromthe group consisting of PLLLLLLSLPPRA (SEQ ID NO:2) and PLLLLLLALPQRA(SEQ ID NO:6).
 6. A nucleic acid encoding the T-cell epitope peptide ofclaim
 5. 7. A vector comprising the nucleic acid of claim
 6. 8. Anisolated cell comprising the vector of claim
 7. 9. A pharmaceuticalcomposition comprising an isolated T-cell epitope peptide adapted torepress an immune response and a pharmaceutically acceptable carrier orexcipient; wherein said peptide is 13-25 amino acids long and comprisesa sequence of PLLLLLLXLPXRA (SEQ ID NO:5), wherein X is an amino acidand does not have to be the same amino acid in each occurrence in agiven sequence, provided that said peptide does not containPLLLLLLALPQRA (SEQ ID NO:6).
 10. The pharmaceutical composition of claim9, wherein the PLLLLLLXLPXRA sequence is PLLLLLLSLPPRA (SEQ ID NO:2).11. The pharmaceutical composition of claim 9, further comprising atarget antigen.
 12. The pharmaceutical composition of claim 10, furthercomprising a target antigen.
 13. The pharmaceutical composition of claim2, further comprising a target antigen.